The envelope glycoprotein, Env, of HIV/SIV mediates viral attachment and entry. As the major surface antigen, the protein forms trimeric spikes on the surface of mature virion, where the receptor-binding subunit gp120 and the fusion subunit gp41 are noncovalently associated. Membrane fusion is driven by large-scale structural rearrangements of the envelope protein stepwise induced by binding to the viral receptors. Each of these conformational states of Env during the fusion process presents distinct antigenic surfaces. The long-term goal of this study is to define structural correlates of neutralization - that is, to understand which of the states might generate neutralizing antibodies against HIV infection. Numerous strategies have failed to produce immunogens capable of eliciting broadly neutralizing antibodies, which are also very rare in HIV infected patients. No one has yet designed an immunogen that presents gp41, and hence the epitopes for broadly neutralizing antibodies such as 2F5 and 4E10, in its native conformation. Insights from our recently determined structure of unliganded gp120 have allowed us to create a new construct, designated gp140-DO, by removing the outer domain and the V1V2 variable loop of gp120. This truncated protein is essentially gp41 constrained into its prefusion conformation by the residual inner domain of gp120. The proposed research aims to obtain structural data leading to a model for the intact, prefusion envelope trimer and to characterize gp41 in its prefusion conformation both structurally and immunologically. We will produce HIV and SIV gp140-DO in large quantity, and characterize, crystallize, ultimately determine the structure by x-ray crystallography. We will also investigate whether the gp140-DO proteins elicit improved neutralizing antibody responses as compared with full-length gp140 immunogens. We expect the work to help us define structural correlates of neutralization and to facilitate vaccine development in general. Specific aims are: (i) To characterize biochemically the gp140 trimeric protein with the outer domain of gp120 deleted (gp140-DO) from both SIV and HIV-1, especially from primary isolates, (ii) To crystallize the gp140-DO proteins and determine their crystal structures, (iii) To evaluate immunogenicity of HIV gp140-DO proteins. [unreadable] [unreadable] [unreadable]